The discovery and development of RNAi and CRISPR/Cas9 genetic screening technologies have provided researchers with invaluable tools for wide-scale and rapid genetic screening. A recent theme of our research program has been to develop methodology for efficient application of these screening technologies in immune cell lineages (Li et al (2015) Sci. Rep. 5:9559), and to implement screens in both human and mouse hematopoietic cells to interrogate the mechanistic basis of immune cell responses to pathogenic stimuli (Sun et al (2016) Sci Signal. 9: ra3; Sun et al (2017) Sci. Data. 4:170007; Li et al (2017) Sci. Data. 4:170008). Our efforts are focused on macrophages as they form the first line of defense against numerous bacterial and viral pathogens and characterization of these initial encounters are central to collaborative efforts in the LISB to generate integrated models of host-pathogen interactions. In FY18 we have continued to develop and refine advanced methods for both quantitative measurement of signaling responses in macrophages (Ernst et al (2018) Methods Mol Biol. 1714:67), and for efficient gene perturbation in macrophage cell models, with particular progress made in application of siRNA transfection methods for primary macrophage cells (Lin et al, In preparation). We are also employing a Kinase Translocation Reporter (KTR) system for each subtype of the MAPKs (Erk, p38 and Jnk) using spectrally distinct fluorescent proteins to permit the simultaneous dynamic measurement of activation of all three MAPK branches in macrophages. This technology is being introduced into mouse and human macrophage cell lines using lentiviruses and also into mouse models using targeted recombination at the Rosa26 safe harbor locus. This approach is already working well in cell lines and promises to uncover a new appreciation of how MAPK dynamics are regulated in macrophages during inflammatory responses. RNAi screen data are susceptible to a myriad of experimental biases, some of which can be mitigated by computational analysis for which we have previously developed sophisticated software tools (Dutta et al (2016) Nat. Commun. 7: 10578). In FY2018, we have further extended this work to develop a novel bioinformatic method termed TRIAGE (Throughput Ranking from Iterative Analysis of Genomic Enrichment). This model applies multiple pathway and network enrichment steps on a screening dataset in an iterative manner, correcting for the biases of individual steps in a complementary fashion. To test and validate this approach, we used the primary and secondary screen data from our completed human and mouse screens of the LPS response (Sun et al (2017) Sci. Data. 4:170007; Li et al (2017) Sci. Data. 4:170008). We compared TRIAGE to current analysis methods for; 1) ability to identify true positives at higher rates 2) overlap rate between orthogonal screens of similar biology 3) overlap rate between RNAi and CRISPR/Cas9 screens and 4) identification rate of canonical TLR pathway genes. In all of these cases, TRIAGE significantly outperformed established analysis methods. We have developed a sophisticated web-based interface for the TRIAGE application, to permit its use by other investigators. A manuscript describing the development and application of the TRIAGE application is in preparation. The signaling pathways and transcription factor responses induced in macrophages upon TLR stimulation are regulated by feedback loops that modulate the kinetics and magnitude of gene transcription. Among these, NF-kB has been a paradigm for a signal- responsive transcription factor that operates in a feedback regulatory network. We have previously described the use of our screening NF-kB reporter cells to identify a novel positive feedback loop in the macrophage NF-kB activation process, which supports a robust inflammatory program at higher TLR ligand doses. Using genome-wide siRNA screen data, we discovered an important role for the transcription factor Ikaros in supporting this inflammatory response (Sung et al (2014) Sci Signal, 7: ra6). In FY18, we published a study that provides further insight into the function and mechanisms of Ikaros action. Using comprehensive genomic analysis of Ikaros-dependent transcription, DNA binding, and chromatin accessibility, we describe unexpected dual repressor and activator functions for Ikaros in the LPS response of murine macrophages. Consistent with the described function of Ikaros as transcriptional repressor, Ikzf1-/- macrophages showed enhanced induction for select responses. In contrast, we observed a dramatic defect in expression of many delayed LPS response genes and ChIP-seq analyses support a key role for Ikaros in sustained NF-kB chromatin binding. Decreased Ikaros expression in Ikzf1+/- mice and human cells dampens these Ikaros-enhanced inflammatory responses, highlighting the importance of quantitative control of Ikaros protein level for its activator function. In the absence of Ikaros, a constitutively open chromatin state was coincident with dysregulation of LPS-induced chromatin remodeling, gene expression, and cytokine responses. Together, our data suggest a central role for Ikaros in coordinating the complex macrophage transcriptional program in response to pathogen challenge. Beyond our continued study of the TLR4 pathway response to bacterial LPS, we are also extending our studies to interrogate the recently discovered cytosolic LPS sensing pathway, which activates the non-canonical inflammasome response and the release of IL-1 family inflammatory cytokines. Recent studies have shown this to be a critical component of the broader physiological response to LPS and a major contributor towards septic shock outcomes in Gram-negative bacterial infections. In FY18, applying our robust siRNA delivery methods for mouse macrophages, we have collaborated with the NIH-NCATS screening facility to complete the secondary phase of a genome-scale screen of the IL-1 alpha response to cytosolic LPS and we are actively studying the gene hits that have emerged from this project. We find that the canonical hit rate for known components of this pathway is particularly high in this dataset, suggesting robust screen assay design and high potential for identifying novel regulators of this critical inflammatory pathway. We also noted a significant enrichment of mitochondrial-associated genes in the screen, supporting an important role for the mitochondria and cellular metabolism in inflammasome activation. Among these genes we identified three nucleotide diphosphate kinases, and we further investigated the role of the Nme4 gene in inflammasome activation. We find that Nme4-/- RAW264.7 cells have a dramatic defect in their IL-1 alpha response to cytosolic LPS. They exhibit constitutively elevated cardiolipin levels in their mitochondrial outer membrane and show defective cardiolipin switching in response to mitochondrial stress signals. Interestingly, we find that Nme4-/- cells have a marked defect in the priming step of inflammasome activation, with a large majority of priming-induced transcriptional increases diminished in the absence of Nme4. Metabolic analysis suggests that Nme4 is critical to the glycolytic commitment induced during inflammasome priming, however we observe normal NF-kB and MAPK activation in primed Nme4-/- cells, suggesting that the mitochondrial and metabolic contribution to inflammasome priming occurs independently of these signaling responses. We also find that Nme4 deficient mice show substantial resistance to LPS-induced endotoxic shock. In ongoing studies, we are using dynamic live cell imaging reporters for mitochondrial function and inflammasome triggering, to further delineate the mitochondrial and metabolic processes that support inflammasome activation.